The ribosome is a complex ribonucleoprotein (RNP) responsible for the essential process of protein synthesis in all living organisms. Assembly of ribosomes involves the transcription and processing of ribosomal RNAs (rRNAs), association of ribosomal proteins, and the activities of many assembly factors. Defects in the ribosome assembly process are associated with changes in virulence and drug resistance in various human pathogens. Alterations in rRNA maturation and thus with associated nucleases, which are involved in this process, have been implicated as being very important for pathogenicity. A clear understanding of this biogenesis cascade will be needed for the development of novel antimicrobial drugs. However, many questions remain regarding the mechanism of rRNA maturation and exactly how this process fits into the ribosome biogenesis cascade. To address these questions the Culver laboratory has developed a novel affinity purification technique to isolate pre-SSU assembly intermediates from E. coli. Purified pre-SSUs from wild-type E. coli contain an precursor 17S rRNA that upon incubation with cell extracts or purified rRNA processing enzymes is processed into mature 16S rRNA. Thus these data suggest that purified RNPs are on- pathway assembly intermediates that can undergo maturation in vitro. Therefore, this proposal seeks to determine how rRNA processing is integrated into small ribosomal subunit (SSU) biogenesis and to decipher the complete mechanism of SSU 16S rRNA maturation in E. coli using this affinity purification method. Aim1 of this proposal is to isolate nd characterize pre-SSU intermediates from mutant E. coli strains perturbed for SSU biogenesis in which distinct rRNA processing intermediates accumulate. Further characterization of these intermediates will divulge the composition of in vivo formed RNPs that are the substrates for rRNA processing and reveal critical assembly events that occur downstream of rRNA cleavage. Secondly, using these purified pre-SSUs as substrates, Aim 2 of this proposal will focus on using cell extracts and purified rRNA processing enzymes to kinetically characterize the molecular mechanism of 16S rRNA maturation in vitro. These data will serve as a foundation for understanding the flux of rRNA processing pathways under various conditions and allow the individual roles of rRNA maturation enzymes to be dissected. Taken together these aims seek to provide an unprecedented glimpse into the mechanism of 16S rRNA maturation and SSU biogenesis in E. coli.